WO2012098100A1 - Vecteurs d'expression contenant des éléments s/mar, cellule et lignées cellulaires et procédés d'utilisation associés - Google Patents

Vecteurs d'expression contenant des éléments s/mar, cellule et lignées cellulaires et procédés d'utilisation associés Download PDF

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WO2012098100A1
WO2012098100A1 PCT/EP2012/050616 EP2012050616W WO2012098100A1 WO 2012098100 A1 WO2012098100 A1 WO 2012098100A1 EP 2012050616 W EP2012050616 W EP 2012050616W WO 2012098100 A1 WO2012098100 A1 WO 2012098100A1
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interest
molecule
region
nucleic acid
mar
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Manfred Wirth
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Helmholtz-Zentrum für Infektionsforschung GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island

Definitions

  • the present invention relates generally to newly discovered S/MAR elements useful in expression vectors.
  • the present invention relates to the use of the MUR-region of CMV as S/MAR element in expression vectors.
  • the present invention relates to vector nucleic acids containing the same as well as host or host cells containing said vector nucleic acids either stably integrated into the host genome or episomally replicating, or alterna- tively, present as a minicircle expression vector for stable or transient expression.
  • the present invention relates to a method for the production of a molecule of interest based on the MUR-region of CMV as a S/MAR region as well as kits or systems containing the vector nucleic acid or cell or cell line according to the present invention.
  • Gene transfer technology in mammalian cells are diverse and include various approaches including in vitro expression in cells engineered for production of proteins of pharmaceutical/industrial interest or with altered metabolism and in vivo expression by gene transfer.
  • viral vectors are the most sufficient vehicles for gene transfer, the use is restricted by major limiting factors, such as the occurrence of immune responses to viral components and interference with cellular integrity.
  • prototypes are found in viral systems which rely on origins of replication (oris), e.g. described from the SV40, BK virus, BPV-1 or EBV. The function of oris is supported by elements mediating retention in the nucleus of the host cell.
  • transient gene expression is an increasingly applied method for rapid production of small amounts of pharmaceutical proteins, especially for evaluation purposes and toxicity testing, without the need to establish a new cell line.
  • the genetic material needed for expression of a target protein is incorporated into the cell's nucleus while avoiding its integra- tion into the genome.
  • the transfer is affected by the help of viral vectors or alternatively, the use of non-viral vectors, such as polyethylenimine.
  • S/MAR scaffold or matrix- attached regions
  • the minicircles described in the art are constructs of approximately 4 kb size established by excision of prokaryotic parts with only one remaining active transcription unit. Minicircles utilize the replication machinery of the host cell due to the association with the nuclear matrix and are not affected by epigenetic silencing.
  • the human cytomegalomovirus (CMV) immediate early promoter/enhancer is known since 1 985 for use in the expression of genes in mammalian cells or cell lines. This promoter/enhancer shows high transcription efficiency and is applied in different approaches for protein production in mammalian cells.
  • the CMV immediate early promoter/enhancer is a 600 bp element consisting of the promoter and enhancer of CMV.
  • a modulator region M, -1 1 45 to -780, and a so called unique region (UR), -780 to -61 0, whereby + 1 is the transcription starting point, e.g. see fig- ure 1 .
  • this MU R region is effective as a bidirectional regulator of gene expression of the immediate early genes of CMV and the open reading frame UL1 27 in opposite direction.
  • the unique region contains binding sides for various transcription factors, like NF1 , CTP, SATPB1 , Fox A, or suppressor of hairy wing. This region mediates repression of transcription of the upstream UL1 27 open reading frame.
  • the modulator is responsible for different expression in differentiated and non-differentiated cells. It has been demonstrated so far that the M or UR region in cell systems for protein expression based on transient or stable gene expression influence of M or UR is not given or very low.
  • S/MAR elements mediate site independent expression in stably transfected cells since these elements protect transcription units integrated into the genome of the cells from chromosomal environment.
  • S/MARs represents borderlines for active and inactive chromatin.
  • S/MARs do not influence transient expression.
  • Broil et. al. describes minicircle performance depending on S/MAR nuclear matrix interactions in CHO expression systems showing that maintenance and stable expression depends on the cell cycle phase during gene transfer. In minicircles S/MAR mediate attachment to the nuclear matrix in sta- ble clones and, thus, mediate maintenance in the nucleus. Further, episomal replication and enhanced stable expression also in the absence of a selection marker are known.
  • An object of the present invention is to provide new vector nucleic acid molecules allowing transient as well as stable expression of molecules of interest in host cells.
  • a need for new elements allowing to increase gene expression in stable transfected cells as well as allowing to influence transient gene expression, in particular, when using episomal minicircles.
  • the present invention relates to the use of the MU R-region of CMV as S/MAR elements in expression vectors. That is, the present inventors recognized that the MU R-region (modulator and unique region) of CMV (cytomegalovirus) acts as S/MAR element in expression vectors suitable both for transient as well as stable expression. In particular, the present inventors recognized that the MU R of rodent, simian or human CMV, in particular, human CMV region represents a new S/MAR element. Thus, it is possible to increase and extend productivity of transient expression when placed into a specific vector location as well as to increase productivity in stable transfection.
  • CMV cytomegalovirus
  • the MU R-region is the nucleic acid sequence of Seq. lD No. 1 .
  • the present invention relates to a vector nucleic acid suitable for expressing at least one molecule of interest in a mammalian cell, comprising the S/MAR-region consisting of the MU R-region of CMV, a promoter and the molecule of interest or a gene encoding the molecule of interest.
  • this vector nucleic acid allows to increase expression of the molecule of interest in stably transfected cell lines, in particular, in parental plasmids, when MU R located 5' of the promoter sequence or is flanking the transcription unit as well as extending the transient gene expression in a cell line, in particular in minicircles if MU R is part of the transcription unit.
  • the present invention relates to cells or cell lines containing the vector nucleic acid according to the present invention.
  • Said cell lines are able to express the molecule of interest transiently or stably and may contain said vector of interest integrated in the host genome or as an episomally replicating vector or as a minicircle expression vector.
  • the present invention relates to a method for the production of a molecule of interest, in particular, a gene of interest or a polypeptide of interest comprising the step of transfecting a cell or cell line, preferably, a mammalian cell or cell line, with an expression vector containing the MU R-region of CMV as S/MAR region.
  • said vector is a vector nucleic acid according to the present invention.
  • the present invention relates to a kit or system containing the vector nucleic acid according to the present invention or a cell or cell line according to the present invention.
  • Said kit or system is particularly useful in the production of recombinant molecules of interest, in particular, nucleic acid molecules of interest or polypeptides of interest.
  • the M + U R guides the transcription of the UL1 27 open reading frame upstream and influences the gene expression of the immediate early genes downstream
  • Figure 2 Relative gene expression in transiently and stably transfected animal cell lines of expression vectors carrying or devoid of an upstream MUR.
  • FIG. 3 Parental plasmids as precursors for the generation of MUR- minicircles.
  • BB bacterial backbone, dashed.
  • the eukaryotic transcription unit consisting of promoter with 5' regulatory upstream region (e.g. the hCMVIE or SV40 promoter preceded by a MUR) a gene of interest (GOI), a poly A signal and a MUR located between GOI and pA within the transcribed region.
  • BB and eukaryotic unit are separated by two FRT sites (not shown), representing the recognition target for yeast FLP recombinase.
  • FIG. 4 Schematic drawing of a minicircle derived from recombination of the parental plasmid after expression of FLP recombinase and purification.
  • the MC consists of the eukaryotic transcription unit including the transcribed MUR. Transcription is thought to allow opening of the MU R double strand to exert the S/MAR activities.
  • FIG. 5 Analysis of stress induced duplex destabilization (SIDD) (Benham et al. , 1 997, J. Mol Biol. , 274; 1 81 -1 96) identifies S/MARs in the upstream region of various animal cytomegaloviruses.
  • SIDD stress induced duplex destabilization
  • the present inventors recognized that the MUR-region (modulator and unique region) of CMV (cytomegaloma virus) has S/MAR element properties. That is, the present inventors recognized that the MUR-region of CMV is able to influence stable as well as transient expression of molecules of interest in cell or cell lines wherein said MUR region is used as S/MAR elements in vector nucleic acid molecules. Hence, in a first aspect, the present invention relates to the use of the MUR- region of CMV as S/MAR element in expression vectors.
  • said use is directed to expression vectors for transient expression or, alternatively, for stable integration into the host chromosome and/or stable expression upon extrachromosonal replication.
  • the S/MAR is a MU R-region of rodent, simian or human CMV.
  • the S/MAR region is the MUR-region of human CMV, like the MUR-region according to Seq. lD NO. 1 .
  • GenBank X03922 entry, -1 145 to -61 0 shows the MUR-region of human CMV, of course, other hCMV related isolates exists containing variations, like substitutions, deletions or additions in the sequence. It is contemplated that said variations in the MU R-region are within the meaning of the term "MU R-region of CMV".
  • the MU R-region useful as S/MAR is derived from simian CMV, e.g. as described in genbank Acc. No. U38308.1 , position 3584-4270; MUR of mouse CMV, Genbank Acc. No. L06570, position 1 -601 ; rat CMV e.g as described in Genbank Acc. No. U62396, postion 1 -721 ; or guinea pig e.g. as described in Genbank Acc. No. FJ355434, position 1 95520-1 94801 .
  • the term MU R-region refers to the modular and unique region of CMV.
  • An example of said region is the nucleic acid sequence of Seq. lD No. 1 representing the MU R-region of human CMV.
  • Another preferred embodiment is the MUR region of simian CMV (Seq. ID. No. 2), mouse CMV (Seq. ID No. 3), rat CMV (Seq. ID. No. 4) and guinea pig CMV (Seq. ID No. 5).
  • S/MAR element stands for scaffold/matrix attach- ment region and refers to DNA sequences conferring anchorage to the nuclear matrix of animal or plant cells. Synonymously the terms “MAR” and “SAR” are known for this element.
  • molecule of interest refers to molecules, either on nucleic acid level or amino acid level.
  • the terms “comprising”, “comprises” and “comprised of” are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms “comprising”, “comprises”, and “comprised of” as well as “including”, “includes”, or “containing”, “contains” as used herein comprise the terms “consisting of", “consists” and “consists of”. All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.
  • a vector nucleic acid suitable for expressing at least molecule of interest in a mammalian cell comprising
  • S/MAR region consisting of the MUR-region of CMV, in particular, of rodent, simian or human CMV, particularly preferred of human CMV, a promoter and
  • the molecule of interest or a gene encoding the molecule of interest is provided.
  • the promoter is not derived from CMV.
  • Said vector nucleic acid is suitable for transient as well as stable expression of the molecule of interest.
  • the vector nucleic acid according to the present invention is a nucleic acid having the following order from the 5' end to the 3' end:
  • S/MAR may be present either at the 5' end or 3' end or both ends.
  • promoter gene encoding the molecule of interest or the molecule of interest, poly A, 3'-S/MAR.
  • the promoter is operably linked with the gene encoding the molecule of interest or the molecule of interest and the poly A tail is operably linked with the gene encoding the molecule of interest or the molecule of interest as well.
  • the vector nucleic acid according to the embodiment described above is particularly suitable for stable expression of the molecule of interest.
  • the vector nucleic acid is a vector nucleic acid wherein the S/MAR region consisting of the MUR-region of CMV is present on both, the 5' end and the 3' end.
  • the vector nucleic acid has the following order from the 5' end to the 3' end: 5'-Promoter operably linked with the molecule of interest or the gene encoding the molecule of interest, S/MAR consisting of the MUR-region of CMV according to the present invention and the poly A tail, wherein the S/MAR and the poly A tail are operably linked with each other.
  • a further MUR region may be present, preferably, derived from CMV.
  • the promoter/enhancer of the vector nucleic acid may be derived from any known and suitable promoter/enhancer described in the art.
  • the promoter/enhancer region is derived from SV40, CMV, ubiquitin C, or
  • the promoter is not CMV.
  • the vector nucleic acid may contain prokaryotic parts. That is, the vec- tor nucleic acids according to the present invention encompass constructs further containing prokaryotic elements allowing for amplification in a host, e.g. a prokaryotic host. These prokaryotic elements may be excised by suitable means in the transfected host to create so called minicircles composed of the vector nucleic acid containing the molecule of interest together with the MUR- region having a S/MAR functionality. Suitable systems are described e.g. in Nehlsen K., et al., 2006, Gene Ther. Mol. Biol, 1 0, 233-244.
  • the vector nucleic acid according to the embodiment of a vector nucleic acid having the order from the 5' end to the 3' end of 5'-promoter, a molecule of in- terest or a gene encoding the molecule of interest, S/MAR consisting of the MUR-region of CMV and poly A, is particularly useful for transient expression as well as for stable expression.
  • the transcribed region comprises the promoter region, the gene of interest region, the S/MAR and the poly A tail.
  • This vector nucleic acid is particularly useful for forming minicircle expression vec- tors for transient expression.
  • this vector nucleic acid also allows stable integration into the host genome or episomal replication.
  • the vector nucleic acid according to the present invention is particularly a vec- tor nucleic acid wherein the molecule of interest is a gene encoding a poly peptide of interest, a gene encoding an antisense or silencer nucleic acid molecule, in particular, an RNA molecule, an antibody, a DNA vaccine, a regulatory RNA molecule, or a non-coding RNA molecule.
  • the present invention provides the use of the MU R-region of CMV, in particular, of rodent, simian or human CMV, particularly preferred of human CMV, as S/MAR element, a vector nucleic acid according to the present invention, a cell or cell line according to the present invention for the expression of a molecule of interest, in particular, a nucleic acid molecule of interest or polypeptide of interest.
  • the present invention provides a method for the production of a molecule of interest, in particular, a gene of interest or a polypeptide of interest.
  • Said method comprises the step of transfecting a cell or cell line, preferably a mammalian cells or cell lines, with an expression vector containing the MU R-region of CMV.
  • the expression vector containing the MU R-region of CMV contains a MU R-region of rodent, simian or human CMV, particularly preferred of the human CMV as S/MAR region.
  • the method is a method using a vector nucleic acid according to the present invention containing the MUR-region of CMV as S/MAR regions.
  • the method according to the present invention allows for the transient expression of a molecule of interest, wherein the expression vector is a vector nucleic acid of the following order: 5'-promoter, a molecule of interest or a gene encoding the molecule of interest, S/MAR consisting of the MU R-region of CMV, poly A wherein the promoter, the molecule of interest or the gene encoding the molecule of interest, S/MAR and poly A operably linked with each other.
  • the method allows for the stable expression of a molecule of interest, wherein the expression vector is a vector nucleic acid as defined herein.
  • the cell or cell line is transfected with the vector nucleic acid and the vector nucleic acid is either integrated to the host genome or is episomally replicating. Alter- natively, a minicircle expression vector is formed in the cell or cell line allowing stable expression of the molecule of interest. It is preferred that for stable expression of the molecule of interest, the vector has the order: 5'-S/MAR, promoter, molecule of interest or nucleic acid encoding the same. In one embodiment thereof, the promoter is not a CMV promoter.
  • the present invention relates to a kit or system containing the vector nucleic acid, according to the present invention or a cell or cell line according to the present invention.
  • the kit or system according to the present invention is particularly useful for the production of recombinant molecules of interest, in particular, nucleic acid molecules of interest or polypeptides of interest.
  • the kit allows for transient or stable expression of said molecules of interest which is particularly desired in pharmaceutical and industrial industry.
  • hCMV The M and U R of hCMV was cloned upstream of the hCMV immediate early promoter in pCMVLUC, an expression plasmid carrying the hCMV immediate early promoter enhancer driving a firefly luciferase as reporter gene according to known methods. Luciferase expression was quantified 2 d after transfection (transient phase) using a standard luminometric assay and compared to the expression of the transfected construct lacking the MU R. Cotransfer of expression constructs using a neomycin resistance expression vector (pAG60, Col- bere-Garapin, et al., 1 981 J. Mol.
  • the minicircles (MC) harboring a 3' MU R within the transcribed region (CMV), a cellular-S/MAR (M1 8, Broil et al. 201 0, J. Mol. Biol. 395, 950-965) and a MUR-less MC (w/o), and all carrying by eukaryotic pro- moter followed by the eGFP reporter gene, were transfected into human embryo kidney HEK 293 cells and GFP expression (x Mean average) was followed in the transient phase using flow cytometry. While the standard MCs loose gene expression as early as one day after transfection the viral S/MAR (MUR) extends the phase of transient expression at least up to day 4. Data are shown in figure 4.
  • SIDD stress induced duplex destabilisation
  • SIDD was used to investigate the regions upstream of CMVIE enhancer promoters of different animal cytomegaloviruses. Peak values disclose the presence of S/MAR elements in the region 5' oft the immediate early promoter/ enhancers.

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Abstract

De manière générale, cette invention concerne des éléments S/MAR nouvellement découverts, utiles dans des vecteurs d'expression. En particulier, cette invention concerne l'utilisation de la région MUR du CMV en tant qu'élément S/MAR dans des vecteurs d'expression. Selon un autre aspect, cette invention concerne des acides nucléiques de vecteur les contenant ainsi qu'un hôte ou des cellules hôtes contenant lesdits acides nucléiques de vecteur intégrés de manière stable dans le génome hôte ou à réplication épisomique, ou en variante, présents sous la forme de vecteurs d'expression de type minicercle pour une expression stable ou passagère. Selon un autre aspect, cette invention concerne un procédé de production d'une molécule d'intérêt basé sur la région MUR du CMV utilisée en tant que région S/MAR ainsi que des trousses ou des systèmes contenant l'acide nucléique de vecteur ou la cellule ou lignée cellulaire selon la présente invention.
PCT/EP2012/050616 2011-01-17 2012-01-17 Vecteurs d'expression contenant des éléments s/mar, cellule et lignées cellulaires et procédés d'utilisation associés WO2012098100A1 (fr)

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EP11151186A EP2476755A1 (fr) 2011-01-17 2011-01-17 Vecteurs d'expression, cellules et lignées cellulaires contenant S/MAR, ainsi que leurs procédés d'utilisation
EP11151186.1 2011-01-17

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021076965A1 (fr) * 2019-10-17 2021-04-22 Minicircle, Inc. Acides nucléiques codant pour des anticorps neutralisant le vih et leurs utilisations

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3456821B2 (fr) 2017-09-19 2024-01-24 Deutsches Krebsforschungszentrum Vecteurs d'adn non intégrants destinés à la modification génétique de cellules

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004044003A2 (fr) * 2002-11-11 2004-05-27 Aventis Pharma Deutschland Gmbh Utilisation du recepteur de edg-2 dans un modele animal d'insuffisance cardiaque
WO2006079068A2 (fr) * 2005-01-20 2006-07-27 University Of Rochester Compositions et methodes permettant d'etudier et de traiter des maladies et des troubles inflammatoires

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004044003A2 (fr) * 2002-11-11 2004-05-27 Aventis Pharma Deutschland Gmbh Utilisation du recepteur de edg-2 dans un modele animal d'insuffisance cardiaque
WO2006079068A2 (fr) * 2005-01-20 2006-07-27 University Of Rochester Compositions et methodes permettant d'etudier et de traiter des maladies et des troubles inflammatoires

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
BENHAM ET AL., J. MOL BIOL., vol. 274, 1997, pages 181 - 196
BI; BENHAM, BIOINFORMATICS, vol. 20, 2004, pages 1477 - 1479
BROIL ET AL., J. MOL. BIOL., vol. 395, 2010, pages 950 - 965
BROIL S. ET AL., J. MOL. BIOL., vol. 395, 2010, pages 950 - 965
BROLL S ET AL: "Minicircle Performance Depending on S/MAR-Nuclear Matrix Interactions", JOURNAL OF MOLECULAR BIOLOGY, vol. 395, no. 5, 5 February 2010 (2010-02-05), LONDON, GB, pages 950 - 965, XP026852116, ISSN: 0022-2836, [retrieved on 20100112] *
BROLL S.V.U.: "Selbstreplizierende nicht-virale Episomen (Minicircles): Expressionseigenschaften und Etablierung im Zellkern", 2009, pages 129PP, XP002642125, Retrieved from the Internet <URL:http://rzbl04.biblio.etc.tu-bs.de:8080/docportal/servlets/MCRFileNodeServlet/DocPortal_derivate_00007710/BrollS_Minicircles_Diss2009.pdf;jsessionid=188CECBCD6449E249D453934647A8F2C> [retrieved on 20110615] *
COL- BERE-GARAPIN ET AL., J. MOL. BIOL., vol. 115, 1981, pages 1 - 14
DATABASE EMBL [online] EBI; 14 November 2006 (2006-11-14), "Murine CMV IE region", XP002642127, Database accession no. L06816 *
DATABASE EMBL [online] EBI; 16 April 2005 (2005-04-16), "Murid (rat) cytomegalovirus IE region", XP002642128, Database accession no. AY166871 *
DATABASE EMBL [online] EBI; 30 July 2009 (2009-07-30), "Caviid (guinea pic) herpesvirus 2 IE region", XP002642129, Database accession no. AB492278 *
DATABASE EMBL [online] EBI; 7 May 2000 (2000-05-07), "Simian CMV IE region", XP002642126, Database accession no. U38308 *
GIROD; MERMOD, NEW COMPREHENSIVE BIOCHEMISTRY, vol. 38, 2003, pages 359 - 379
JIALING LEE ET AL: "Cell-specific effects of human cytomegalovirus unique region on recombinant protein expression", BIOTECHNOLOGY LETTERS, vol. 29, no. 12, 27 July 2007 (2007-07-27), SPRINGER NETHERLANDS, DORDRECHT, pages 1797 - 1802, XP019548730, ISSN: 1573-6776, DOI: 10.1007/S10529-007-9490-7 *
KIM; KIM, J. BIOT., vol. 107, 2004, pages 95 - 105
LEE ET AL: "Cellular homeoproteins, SATB1 and CDP, bind to the unique region between the human cytomegalovirus UL127 and major immediate-early genes", VIROLOGY, vol. 366, no. 1, 29 August 2007 (2007-08-29), ACADEMIC PRESS,ORLANDO, US, pages 117 - 125, XP022232348, ISSN: 0042-6822, DOI: 10.1016/J.VIROL.2007.04.024 *
LUFINO M.M.P., MANSERVIGI R. & WADE-MARTINS R.: "A S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency.", NUCL. ACIDS RES., vol. 35, no. 15, E98, 2 August 2007 (2007-08-02), pages 1 - 10, XP002642131 *
NEHLSEN K. ET AL., GENE THER. MOL. BIOL, vol. 10, 2006, pages 233 - 244
STEHLE I.M. & SCHAARSCHMIDT D.: "Autonom replizierende Vektoren für Säugerzellen", BIOSPEKTRUM, vol. 2, 2005, pages 162 - 165, XP002642130 *
ZAHN-ZABAL; KOBR, J. BIOT., vol. 87, 2001, pages 29 - 42

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WO2021076965A1 (fr) * 2019-10-17 2021-04-22 Minicircle, Inc. Acides nucléiques codant pour des anticorps neutralisant le vih et leurs utilisations

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